Oligonucleotides Related to Lipid Membrane Attachments

ABSTRACT

Oligonucleotide structures are provided that are capable of forming more stable bonds to a lipid membrane and thereby generate an improved control of the process whereby oligonucleotide linkers are introduced to lipid membranes. Methods of forming lipid membrane oligonucleotide attachments are provided including lipid vesicles. The oligonucleotides typically comprise at least two hydrophobic anchoring moieties capable of being attached to a lipid membrane. Said moieties may be attached at the terminal ends of an oligonucleotide or, in the case of a first and second strand forming a duplex, at the same terminal end one of the strands other end not being part of the duplex leaving it free to hybridize to additional strands. The lipid vesicles attached with the oligonucleotide can be used in biosensors and may contain membrane proteins.

BACKGROUND OF THE INVENTION

Rapid progress in protein-chip technologies is today made with respectto water soluble proteins (Kodadek, T., Chemistry and Biology 2001, 8,105-115), but to generate a signature of the whole proteome make-up alsomembrane proteins, which constitute an important group of proteins beinga common target for disease diagnostics and therapeutic drugs, must alsobe addressable. However, this class of proteins are often identified asan extremely difficult group of proteins to be analysed on this format.In fact, the first low-density protein chip based on membrane proteinswas only recently reported (Fang, Y.; Frutos, A. G.; Lahiri, J., Journalof the American Chemical Society 2002, 124, (11), 2394-2395),demonstrating an array produced via micro-dispensing of Gprotein-coupled receptor (GPCR) containing lipid membranes. To fullyexplore the potential of array-based analysis of membrane proteins,tethered lipid vesicles have recently emerged as a most promisingalternative, non-the least since they offer the possibility to measurealso membrane-protein mediated material transport across the membrane(Stamou, D.; Duschl, C.; Delamarche, E.; Vogel, H., AngewandteChemie-International Edition 2003, 42, (45), 5580-5583). Means tocontrol the positioning of different types of vesicles on pre-definedregions are still, to a large extent, lacking. By combining the conceptof DNA-labeled vesicles (Patolsky, F.; Lichtenstein, A.; Willner, I.,Journal of the American Chemical Society 2000, 122, (2), 418-4)previously utilized for signal enhancement of DNA hybridizationdetection (with the concept of using DNA-labeled biomolecules forsite-selective binding on cDNA arrays (Niemeyer, C. M., Science 2002,297, (5578), 62). It has recently been demonstrated by the presentinventors (Svedhem, S.; Pfeiffer, I.; Larsson, C.; Wingren, C.;Borrebaeck, C.; Höök, F., Chem Bio Chem 2003, (4), 339-343) and others(Yoshina-Ishii, C.; Boxer, S. G., Journal of the American ChemicalSociety 2003, 125, (13), 3696-3697) to use low density cDNA arrays forsite-selective and sequence specific coupling of DNA-tagged lipidvesicles. Instead of using covalent coupling of DNA to chemically activelipids (Yoshina-Ishii, C et al and the article Patolsky, F.; Katz, E.;Bardea, A.; Willner, I., Langmuir 1999, 15, (11), 3703-3706), we madeuse of cholesterol-modified ss-DNA for spontaneous anchoring into thehydrophobic interior of lipid membranes. This means of anchoring DNAadds a three-folded advantage. This is so because the method (i) isfaster (tens of minutes compared with hours), (ii) does not requirechemically modified lipids to be introduced and (iii) makes use of anaturally occurring membrane constituent, thus eliminating the risk forside effects induced by chemically reactive lipid head groups onincorporated membrane constituents. However, the cholesterol-basedanchoring of DNA to lipid membranes turns out to be relatively weak,thus complicating quantitative control of the number of DNA pervesicles. In addition, site selective sorting of differently DNA-taggedvesicles to cDNA arrays, must, due to DNA exchange between differentlytagged vesicles, be accomplished in a sequential, rather than parallelmanner (see above recited articles by Svedhem et al Yshina-Ishii et al)

SUMMARY OF INVENTION

The present invention aims to provide oligonucleotide structures thatare capable of forming more stable bonds to a lipid membrane and therebygenerate an improved control of the process whereby oligonucleotidelinkers are introduced to lipid membranes. The invention is alsodirected at methods of forming lipid membrane oligonucleotideattachments and lipid vesicles provided with such oligonucleotides, aswell as methods of forming such vesicles.

DESCRIPTION OF INVENTION

To facilitate an understanding of the present invention, a number ofterms are defined below.

As used herein, the term “vesicle” or “liposome” refers typically tospherical structures (5 nm to 20 μm in diameter) built up by lipidmembranes, which may or may not contain proteins, glycolipids, steroidsor other membrane-associated components. The terms “liposome” and“vesicle” are used interchangeable herein. Vesicles can be naturally (egthe vesicles present in the cytoplasm of cells that transport moleculesand partition specific cellular functions) or synthetically (egliposomes) generated. The term “vesicle” is here also used for“micelles” which are particles comprising of lipids, which particleshave a hydrophilic exterior and a hydrophobic interior.

As used herein, the term “nucleotide” refers to any nucleic acid, suchas DNA and RNA, as well nucleic acid analogues such as, but not limitedto, PNA (Peptide Nucleic Acid), LNA (Locked Nucleic Acid) and Morpholinonucleic acid analogues. The term also relates to any nucleotidecomprising of the known base analogues of DNA and RNA.

As used herein the term “oligonucleotide” refers to a short length of psingle-stranded polynucleotide chain. Oligonucleotides are typicallyless than 100 residues long, however, as used herein, the term is alsointended to encompass longer polynucleotides. The term refers to allcombinations of nucleotides as defined above, forming a polymer ofnucleotides.

As used herein, the term “hybridisation” is used in reference to thepairing of essentially complementary nucleic acids often referred to asWatson-Crick-hybridisation as well as the hybridisation referred to asHoogsteen-hybridisation. As used herein, the term “immobilisation”refers to the attachment or entrapment, either chemically or otherwise,of material to a transducer surface in a manner that confines, but notnecessarily restricts, the movement of the material.

As used herein, the term “analytes” refers to any material that is to beanalysed.

As used herein, the term “biosensors” refers to any sensor device thatis partially or entirely composed of biological molecules. In atraditional sense, the term refers to “an analytical tool or systemconsisting of an immobilised biological material (such as enzyme,antibody, whole cell, organell, or a combination thereof) in intimatecontact with a suitable transducer device which will convert thebiochemical signal into a quantifiable electrical signal” (Gronow,Trends Biochem Sci 9, 336, 1984).

As used herein, the term “multilayer” refers to structures comprised ofat least a second layer formed on top of a first layer. The individuallayers may or may not interact with one another.

As used herein, the term “biologically active compound” refers tobiological compounds that are capable of interacting with other materialor compounds. Such biologically active compounds can include, but arenot limited to, proteins, antibodies, nucleotides, lipids, carbohydratesand combinations thereof. The terms “receptor” and “biologically activecompound” are used interchangeable herein.

As used herein, the term “membrane protein” refers to proteins orpolypeptides, which are connected to or inserted in a lipid membrane ina lipid layer.

As used herein, the term outwardly projecting compound” refers to acompound with a part that is projecting out from a surface. In the casewhere the surface is essentially spherical, as in the case withvesicles, the term means that the compounds project from the surfacetowards the surroundings.

As used herein, the term “surface” shall be used in its widest sense. Itencompasses all compound that can be used a support means on whichstructures can be immobilized.

As used herein, the term “linker adapted for binding” refers to that thelinker comprises a compound with ability to bind to another compound.

As used herein, the term “linker available for binding” refers to that alinker is adapted for binding, but the linker is not yet bound toanother linker, or that all binding sites of the linker are not yetoccupied.

In its most general terms the present invention refers to anoligonucleotide comprising at least two hydrophobic anchoring moietiescapable of being attached to a lipid membrane. The anchoring moietiesserve to bind directly to the lipid membrane by hydrophobic interactionat adjacent sites of the membrane and aim essentially to permanentlyattach the oligonucleotide which may in turn be provided with multiplebiological functions according established techniques and serve may as alinker to build up multilayered surfaces of the type as explained in ourparallel application SE 03 01038-6 and further below.

Also in general terms, the present invention refers to a method offorming a lipid membrane attached linker, wherein an oligonucleotidehaving two or more hydrophobic anchoring moieties contact a lipidmembrane, thereby accomplishing a direct attachment of saidoligonucleotide by said moieties at adjacent sites on the same membrane.Preferably, the membrane forms a lipid vesicle and the membrane is abilayer membrane. The method enables a surprisingly strong coupling ofthe oligonucleotide to the membrane that is practically irreversible.

Preferably, the hydrophobic anchoring moieties are located in theoligonucleotide terminal ends and the lipid membrane is the part oflipid vesicle. In one aspect, the oligonucleotide comprises a firststrand and a second strand of nucleic acid, said two strands beinghybridised to each other in a duplex section in a manner that the firststrand terminal end is not a part of said duplex section and is freefrom a hydrophobic anchoring moiety. Preferably, the hydrophobicanchoring moieties are covalently attached to the adjacent terminal endsof said first and second strands.

In another aspect polyvalent oligonucleotides can be assembled so as toprovide multiple (more than two) attachment points to the lipidmembrane. The present inventors contemplate that the need to increasethe number of hydrophobic anchoring units may occur as a result of usinglonger nucleic acids with higher water solubility. For this purpose, thepresent invention is alternatively directed at oligonucleotidescomprising n additional strands to the first and second strand (n beingan integer and n>0). Each additional strand is provided with a terminalhydrophobic anchoring moiety, wherein a first additional strand ishybridized to said second strand and wherein a second additional strandis hybridized to the first additional strand and strand n is hybridizedto strand n−1.

In still another aspect, the oligonucleotides can be construed toconstitute links to create multiplayer structures including a pluralityof lipid vesicles, or other assemblies useful when designing chemicallyor biologically active surfaces. Such oligonucleotides comprises a firstand a second strand said two strands being hybridized to each other in aduplex region in a manner that leaves the first strand free to hybridizewith a third strand. The free end of the first strand may also includeother agents, such as labelling agent, an antibody, a capturing agentcapable of extracting desirable agents from a surrounding fluid or aconventional agent with specific binding capacity. In one embodiment ofthis aspect, the oligonucleotide will have a first strand withhydrophobic anchoring moieties in both its terminal ends to which stranda third strand with, or without, a terminal hydrophobic anchoring moietycan be hybridized, so first and third strands have adjacent hydrophobicanchoring moieties.

The hydrophobic anchoring moiety is selected among, for example,steroids, fatty acids, hydrophobic peptides and lipids; most preferablythe hydrophobic anchoring moiety is cholesterol or a derivative thereof.

In order to form a suitably flexible structure with an optimumpossibility to associate with the hydrophobic parts of the lipidmembrane, the inventive oligonucleotides have the hydrophobic anchoringmoieties spaced apart from the duplex section by a spacing group or asufficient number of non-hybridized nucleic acid units. In order toobtain oligonucleotide structures with optimal flexibility/rigidity bythe chain length between the duplex section and the chain chemistry canbe modified.

The oligonucleotide can be generally adapted and available to be linkedby specific binding to a surface immobilized linker or to another lipidmembrane attached linker. The linkage can be mediated with nucleic acidhybridisation or by other types of specific binding well understood toskilled persons. For example, the oligonucleotides can comprising asection of peptide nucleic acids (PNA) capable of forming PNA-peptidecomplexes. Alternatively, the oligonucleotides can be immobilizeddirectly to surface, either to a lipid membrane or to another suitablecompound or structure, for example by the free end of the first strand.There are numerous routes to enable surface immobilization of nucleicacids known and available to artisans in this field and no furtherdiscussion is necessary in the present context.

According to a preferred embodiment of the present invention, the firststrand is longer (i.e. includes more nucleic acid units) than the secondstrand. The first and second strands preferably have a duplex regioninvolving the terminal end of the second strand. According to onesuitable example, the first strand has essentially double the amount ofnucleic acid monomers than the second strand, said first and secondstrand have a cholesterol molecule attached to their free 5′ and3′-ends, respectively. According to a specific example, theoligonucleotides have a first strand of a 30mer DNA and the secondstrand of a 15-mer DNA having 12 complementary bases.

The oligonucleotides are preferably to be attached to lipid vesicles.The so formed lipid vesicles can be designed with different additionalfunctionalities. For example such lipid vesicles can containelectrochemically detectable reporter molecules in a manner outlined byWO 02/081739 and WO 02/081738 which both are incorporated as references.The lipid vesicles may include biologically active compounds exhibitingbiological functionality, such as membrane proteins, as discussed inmore detail in the aforementioned SE 0301038-6.

The present invention is further directed at surface immobilizedstructures comprising a plurality of vesicles having membrane attachedoligonucleotides of the above mentioned features. To build up suchstructures, the vesicles are adapted and available to be linked byspecific binding to any of a surface immobilized linker, another lipidvesicle attached linker or to the type of surface immobilizedoligonucleotide mentioned above. The surface immobilized structures cantypically be used in biosensors, but numerous other applications wouldalso be conceivable.

DETAILED AND EXEMPLIFYING PART OF THE DESCRIPTION

FIG. 1 shows a bivalent cholesterol based oligonucleotide and changes incoupled mass from quartz microbalance with dissipation monitoringmeasurements upon stepwise addition different cholesterol-DNAassemblies.

FIG. 2 shows a schematic illustration of the DNA array produced adescribed in the specification and micrographs illustrating the sortingof differently DNA-tagged vesicles.

FIG. 3 shows a schematic example of a polyvalent oligonucleotide withmore than two hydrophobic anchoring moieties available lipid membraneattachment.

By mimicking Natures way of utilizing multivalent interactions wepresent in the present work a novel means of improving the strength ofcholesterol-based DNA coupling to lipid membranes (Mammen, M.; Choi, S.K; Whitesides, G. M., Angewandte Chemie-International Edition 1998, 37,(20), 2755-2794). A bivalent cholesterol-based coupling of DNA wasaccomplished by hybridization between a 15-mer DNA and a 30-mer DNA,being modified with cholesterol in the 3′- and 5′-end, of respectively(FIG. 1).

Water was deionized and filtered (MilliQ unit, Millipore). DNA strands:5′-TAG TTG-TGA-CGT-ACA-CCC-CC-3′ (DNA_(A′));5′-TAT-TTC-TGA-TGT-CCA-CCC-CC-3′ (DNA_(B′));5′-TGT-ACG-TCA-CAA-CTA-CCC-CC-3′ (DNA_(A));5′-TGG-ACA-TCA-GAA-ATA-CCC-CC-3′ (DNA_(B));5′-TAG-TTG-TGA-CGT-ACA-AAG-CAG-GAG-ATC-CCC-3′ (DNA_(C));5′-TAT-TTC-TGA-TGT-CCA-AGC-CAC-GAG-ATC-CCC-3′ (DNA_(D));5′-CCC-GAT-CTC-CTG-CTT-3′ (DNA_(C′)); 5′-CCC-GAA-CTC-GTG-GCT-3′(DNA_(D′)), derivatised at the 3′-end with biotin (biotin-DNA_(B)) orcholesterol (chol-DNA_(A′); chol-DNA_(B); chol-DNA_(B′)) or at the5′-end with cholesterol (chol-DNA_(C), chol-DNA_(C′), chol-DNA_(D),chol-DNA_(D′)) (MedProbe, Norway). Stock solutions of DNA conjugates (20μM in Buffer I: 10 mM Tris, 1 mM EDTA, pH 8.0) and proteins(biotin-labeled BSA (Sigma, 1 mg/mL in water), neutravidin (Pierce, 1mg/mL in Buffer II: 10 mM Tris, pH 8.0, 100 mM NaCl) were aliquoted andstored at −20° C. 1-Palmitoyl-2-Oleoyl-sn-Glycero-3-Phosphocholine(POPC, Avanti Polar Lipids, Ala., USA) was dissolved in chloroform. Forfluorescent vesicles, 0.5% (w/w) of Lissamine™ rhodamine B1,2-dihexadecanoyl-sn-glycero-3-phosphoethanolamine (rhodamine-DHPE)(Molecular Probes, USA) or2-(12-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino)dodecanoyl-1-hexadecanoyl-sn-glycero-3-phosphocholine(NBD-HPC) (Molecular Probes, USA) was added to the lipid solution. Lipidvesicles were prepared by evaporation of the solvent under N₂ (>1 h),followed by hydration in buffer (5 mg/mL) and extrusion through 0.1 and0.03 μm polycarbonate membranes 11× each (Whatman, USA), stored at 4° C.under N₂. DNA-labeling was achieved by addition of 0.5% (w/w) ofchol-DNA to the vesicle solution, corresponding to ˜4 DNA per vesicle.All experiments were made be dissolving the stock solutions in Buffer IIto given concentrations. Substrates (AT-cut quartz crystals, f₀=5 MHz,with either gold or SiO₂) and the QCM-D instrument (Q-sense D 300) werefrom Q-sense AB, Sweden. The crystals were cleaned in 10 mM SDS (>15′),followed by 2× rinsing with water, drying (N₂), and UV-ozone treatment(10′). The microscope used for imaging was a Zeiss Axioplan 2fluorescence microscope. SiO₂-coated crystals were patterned byevaporation of 3 nm of Ti and 100 nm of Au through a mask.]

The detailed design of the construct was defined by choosing 12 bases onthe 30-mer strand to be complementary to 12 bases on 15-mer strand. Thesequences were chosen such that the duplex formed by incubating thesestrands forced the two cholesterol moieties into close proximity, stillseparated from the duplex region by a pair of non-hybridized (3C)spacers.

FIG. 1 a shows changes in f (c.f. coupled mass) from quartz crystalmicrobalance with dissipation monitoring (QCM-D) measurements uponstepwise addition of chol-DNA_(A′) at increasing concentrations to aSPB-coated SiO₂ surface, formed as described previously (CA Keller etal. Biophysical Journ. 75(3), 1397-1402). Temporal variations in fobtained upon addition of (blue) chol-DNA_(C) after spontaneousformation of an SPB (t=2 to 4 min) on a SiO₂-coated QCM sensor surfaceat increasing concentrations: 5, 10, 25, 50, 100 nM at a flow rate of250 μL/min. After saturated binding at 100 nM, the system was thoroughlyrinsed in buffer, demonstrating fully reversible binding in agreementwith a Langmuir-adsorption behavior (due to the fact that waterentrapped in the DNA film is sensed by QCM, see e.g. 14, changes in fcannot be used to quantify the amount of coupled mass, but were used fora relative comparison of coupled mass vs. concentration only) revealingK_(d)(=k_(off)/k_(on)) and k_(off) values of 16.7±4 nM and ˜5.8×10⁻⁴s⁻¹, respectively (addition of the 30-mer chol-DNA_(C) displays kineticssimilar to that of chol-DNA_(A′) and chol-DNA_(B′) (not shown)).

With reference to FIG. 1 b, the same type of data as in the experimentalcontext demonstrated with FIG. 1 a, obtained upon addition of the DNAconstruct comprised of pre-hybridized (30 min incubation) chol-DNA_(C)and chol-DNA_(C′) upon increasing concentration: 25, 37.5, 50 and 75 nMand 5 nM, only. After saturated binding, the solutions were exchanged topure Buffer II. Also shown as an inset is an addition of biotin-DNA_(B),being complementary to 15 free hanging bases on the cholDNA_(C)/chol-DNA_(C′) duplex construct. The binding of the duplexconstruct (chol-DNA_(C)/chol-DNA_(C′)) carrying two cholesterol moietiesdisplays irreversible coupling (see FIG. 1 b) independent onconcentration. This excludes a Langmuir-based analysis of the data, butshows that k_(off) is reduced by at least one order of magnitudecompared with the monovalent coupling. Thus, under the assumption thatk_(on) is similar for the mono- and bivalent coupling (see the aboverecited article by M Mammen et al.) the affinity constant (1/K_(d)) is,at least, one order of magnitude higher for the bivalent coupling. Evenif the increase in the binding strength may very well be larger than so,and even approach the theoretical value of (1/K_(d))², the mostimportant observation is that the coupling is irreversible.

First, this means that the bivalent coupling can be used to preciselycontrol the number of DNA per lipid-membrane area. Second, the rapidbinding upon addition of fully complementary biotin-DNA_(B) (inset inFIG. 2 b), demonstrates the feasibility of this template for detailedDNA-hybridization kinetics studies. Third, exchange of DNA betweendifferently DNA-modified vesicles is likely to be significantly reduced.

To test the latter hypothesis, the bivalent cholesterol coupling wastested by producing biotin-DNA_(B)-modified gold spots surrounded by aplanar SPB modified with chol-DNA_(A), thus comprising the simplestpossible “cDNA array” (FIG. 2).

Referring to FIG. 2 (Left) showing a schematic illustration of the DNAarray produced as described previously (see the above article by Svedhemet al) In brief, the surface pattern was modified by preferentialadsorption of biotin-BSA (10 μγ/μL) to the Au spots over the surroundingSiO₂, followed by addition of POPC lipid vesicle solution (20microgram/mL), rendering the surrounding SiO₂ substrate modified with anSPB whereas only weak adsorption occur to the biotin-BSA modified goldspots (Svedhem et al.). This was then followed by subsequent additionsof (i) neutravidin (10.0 microgram/mL), biotin-DNA_(B) (0.1 microM) andchol-DNA_(A) (0.1 microM). FIG. 2 (Right) shows micrographs (i-iv)illustrating the sorting of differently DNA-tagged vesicles obtained byexposing the DNA-modified substrate to a mixture of Rhodamine-labeledvesicles (exc.=550 nm/em.=590 nm) and NBD-labeled vesicles (exc.=460nm/em.=550 nm) being modified with: chol-DNA_(A′) and chol-DNA_(B′),respectively (micrographs i and ii), and bivalently-coupled DNAconstructs comprised of chol-DNA_(C)/chol-DNA_(C′) andchol-DNA_(D)/chol-DNA_(D′), respectively. The DNA concentration wasadjusted to ˜4 DNA per vesicle. The vesicle suspensions were incubatedfor five minutes prior to exposure, and analyzed after 30 min with agreen filter (exc.=450-490 nm/em.=515-565 nm) for image ii) and iv) anda red filter (exc.=546 nm/em.=590 nm) for image i) and ii).

To evaluate parallel sorting from a mixture of two types of vesicles,the “cDNA array” was exposed to two types of vesicle suspensions. Onecontained two differently fluorescent labeled vesicles (red and green)being modified with chol-DNA_(A′) and chol-DNA_(B′), respectively, (c.f.FIG. 1 a). The other contained the same types of vesicles beingseparately modified via bivalently coupled DNA constructs, carryingsingle stranded regions complementary to the immobilize chol-DNA_(A) andbiotin-DNA_(B), respectively (c.f. inset in FIG. 1 b). Indeed, thevesicle suspension containing vesicles tagged with the bivalentlycoupled DNA demonstrates sequence specific and site selective binding tothe predefined regions on the surface (iii. and iv in FIG. 2), whereasthe monovalently modified vesicles appears to be distributed on bothregions (i. and ii. in FIG. 2). The over all lower fluorescence on theSPB substrate is attributed to the lower coverage of chol-DNA_(B) thanbiotin-DNA_(A), and the weak fluorescent dots on the spot in image iv.)is attributed to chol-DNA_(A) binding to non-specifically adsorbed lipidvesicles to biotin-BSA during the SPB formation process (see FigureLegend).

Even if the strength of the bivalent cholesterol-based coupling must notnecessarily by higher than that obtained upon covalent coupling to anactivated lipid head group, we emphasize the simplicity of the principleand its broad application areas, including a large variety of lipidassemblies, such as, for example, lipid vesicles produced by cells orformed from crude cell membranes (to be published). Furthermore, thesuccessful use of a DNA-modified SPB for hybridization detection undercontrolled flow conditions (FIG. 1 b), points towards an interestingtemplate for drug-, protein- and DNA-DNA interaction studies. This is inparticular so, since the DNA coverage can be precisely controlled, whichis known to be critical in the case of immobilized DNA (Larsson, C.;Rodahl, M.; Hook, F., Analytical Chemistry 2003, 75, (19), 5080-5087 andShchepinov, M. S.; CaseGreen, S. C.; Southern, E. M., Nucleic AcidsResearch 1997, 25, (6), 1155-1161). Furthermore, the commonly usedstreptavidin templates used for these purposes may in certain casesinduce unwanted non-specific protein binding, which is likely to besignificantly reduced in this case. Finally, our means of utilizing DNAas a building block to construct a bivalent coupling is easily extendedto DNA constructs rendering multivalent interactions, thus comprising asimple model system for fundamental studies to support, for example,recent theoretical development in this field (Kitov, P. I.; Bundle, D.R., Journal of the American Chemical Society 2003, 125, (52),16271-16284).

1. An oligonucleotide comprising at least two hydrophobic anchoringmoieties capable of being attached to a lipid membrane.
 2. Anoligonucleotide according to claim 1, wherein said hydrophobic anchoringmoieties are located in its terminal ends.
 3. An oligonucleotideaccording to claim 2 comprising a first strand and a second strand ofnucleic acid, said two strands being hybridised to each other in aduplex section in a manner that a first strand terminal end is not apart of said duplex section and free from a hydrophobic anchoringmoiety.
 4. An oligonucleotide according to claim 2, wherein twohydrophobic anchoring moieties are covalently attached to the adjacentterminal ends of said first and second strands.
 5. An oligonucleotideaccording to claim 3 comprising n additional strands; n being an integerand n>0; wherein the additional strands are each provided with aterminal hydrophobic anchoring moiety, wherein a first additional strandis hybridized to said second strand and wherein a second additionalstrand is hybridized to the first additional strand and strand n ishybridized to strand n−1.
 6. An oligonucleotide according to claim 2comprising a first and a second strand said two strands being hybridizedto each other in a duplex region in a manner that leaves the firststrand free to hybridize with a third strand.
 7. An oligonucleotideaccording to claim 6, wherein said first strand has hydrophobicanchoring moieties in both terminal ends.
 8. An oligonucleotideaccording to claim 7, wherein said third strand has a terminalhydrophobic anchoring moiety so first and third strands have adjacenthydrophobic anchoring moieties.
 9. An oligonucleotide according to claim1, wherein the hydrophobic anchoring moiety is selected among steroids,fatty acids, hydrophobic peptides and lipids.
 10. An oligonucleotideaccording to claim 9, wherein the hydrophobic anchoring moiety ischolesterol or a derivative thereof.
 11. An oligonucleotide according toclaim 3, wherein the hydrophobic anchoring moiety is spaced apart fromthe duplex section by a spacing group or a sufficient number ofnon-hybridized nucleic acid units.
 12. An oligonucleotide according toclaim 1 adapted and available to be linked by specific binding to asurface immobilized linker or to another lipid membrane attached linker.13. An oligonucleotide according to claim 1 immobilized to a surface.14. An oligonucleotide according to claim 2, wherein the first strand islonger than the second strand, said first and second strands have aduplex region involving the terminal end of the second strand.
 15. Anoligonucleotide according to claim 8, wherein the first strand hasessentially double the amount of nucleic acid monomers than the secondstrand, said first and second strand have a cholesterol moleculeattached to their free 5′ and 3′-ends, respectively.
 16. Anoligonucleotide according to claim 1 comprising a section of peptidenucleic acids (PNA) capable of forming PNA-peptide complexes.
 17. Anoligonucleotide according to claim 9, wherein the first strand is 30-merDNA; the second strand is a 15-mer DNA having 12 complementary bases.18. A lipid vesicle comprising an oligonucleotide according to claim 1attached to its lipid membrane.
 19. A lipid vesicle according to claim18 comprising electrochemically detectable reporter molecules.
 20. Alipid vesicle according to claim 18 comprising biologically activecompounds exhibiting biological functionality.
 21. A lipid vesicleaccording to claim 20, wherein said biologically active compound is amembrane protein.
 22. A surface immobilized structure comprising aplurality of vesicles according to claim 18, wherein said vesicles areadapted and available to be linked by specific binding to any of asurface immobilized linker, another lipid vesicle attached linker or asurface immobilized oligonucleotide comprising at least two hydrophobicanchoring moieties capable of being attached to a lipid membrane.
 23. Abiosensor including a surface immobilized oligonucleotide according toclaim
 13. 24. A method of forming a lipid membrane attached linker,wherein an oligonucleotide according to claim 1 having two or morehydrophobic anchoring moieties contacts a lipid membrane, therebyaccomplishing a direct attachment of said oligonucleotide by saidmoieties at adjacent sites on the same membrane.
 25. A method accordingto claim 24, wherein said membrane forms a lipid vesicle.
 26. A methodaccording to claim 24 wherein said membrane is a bilayer membrane.
 27. Amethod according to claim 24, wherein said attachment is irreversible.